The HARC Center has made rapid progress in systematically characterizing physical interactions between HIV proteins and host cell pathways using proteomic approaches. The functional roles of these emerging pathways now need to be experimentally tested, which requires targeted genome engineering in primary human T cells. CRISPR-Cas9 is a revolutionary technology that enables re-writing human genetic information in a programmable, site-specific manner. Moreover, the HARC Center recently overcame longstanding hurdles in performing genome editing in primary human T cells. We achieved highly efficient knock-out and knock-in genome engineering in human CD4+ T cells using Cas9:guide RNA ribonucleoproteins (Cas9 RNPs). The ability to precisely replace DNA sequences in the genome of functional T cells now provides unprecedented opportunities to test the mechanistic effects of specific host and viral genetic factors in HIV pathogenesis. Our new tools for genetic manipulation of human T cells strongly complement the HARC Center?s strengths in structural biology and proteomics. We now propose to expand our ongoing dissection of the mechanistic effects of host factors in primary human T cells. These systematic efforts will identify pathways regulated by HIV-interacting factors and functionally test their roles in HIV infection. These studies offer novel means to integrate human genetics and genome engineering with the existing strengths of the HARC Center. Cas9 RNPs will not only allow gene ablation, but will also allow us to edit specific domains or even replace individual amino acids. We have a particular interest in the functional and structural consequences of naturally occurring genetic variants associated with resistance to HIV or delayed progression to AIDS. Finally, Cas9 RNP genome engineering will allow affinity tagging of endogenous host factors in primary cells to enable unprecedented studies of macromolecular complexes involving human and HIV proteins. Collectively, these genetic studies will strengthen the central HARC mission to understand interactions between HIV viral proteins and host molecules and should open new experimental and therapeutic avenues for the broader HIV research community.